382
Journal of Health Science, 54(4) 382–389 (2008)
Effects of Exposure to Decabromodiphenyl Ether on the
Development of the Immune System in Rats
Reiko Teshima,∗ Ryosuke Nakamura, Rika Nakamura, Akiko Hachisuka,
Jun-ichi Sawada, and Makoto Shibutani1
Division of Biochemistry and Immunochemistry, National Institute of Health Sciences, 1–18–1 Kamiyoga, Setagaya-ku, Tokyo 158–
8501, Japan
(Received September 30, 2007; Accepted May 1, 2008)
To evaluate the developmental toxicity of decabromodiphenylether (DBDE) after exposure during the period
from late gestation to after lactation, maternal Sprague-Dawley rats were given DBDE at dietary concentrations
of 0, 10, 100, and 1000 ppm from gestational day 10 (GD 10) to postnatal day (PND) 21. On PND 21 and 77,
lymphocytes (Lymph) in the spleen, thymus, and peripheral blood of male pups were subjected to flow cytometric
analyses for expression of surface markers [CD3, CD4, CD8a, CD25, CD45RA, CD71, and CD161(NKRP1A)].
On PND 21, the proportions of splenic CD4+ T cells in the 10-ppm group, activated B (CD45RA+CD71+) cells
in the 100- to 1000-ppm groups, and activated T cells (CD3+CD71+) in the 1000-ppm group were significantly
decreased, and the population of peripheral CD161+ natural kiler cells on PND 21 and 77 had decreased in the 100to 1000-ppm groups. In the 1000-ppm group, the serum T3 level was significantly decreased on PND 21 and the
serum T4 level was decreased on PND 77. The body, spleen, and thymus weights were not significantly decreased,
but liver weight was significantly increased on PND 21 in the 10- to 1000-ppm groups. These results suggest that
on PND 21, developmental exposure to the highest dose of DBDE had a weak immunomodulatory effect. Although
the most of the immunomodulatory effect had recovered to normal levels on PND 77, a decreasing effect on the
natural killer (NK) cell population remained.
Key words —— decabromodiphenyl ether, immune system, developmental exposure, natural killer cells
INTRODUCTION
Brominated flame retardants (BFRs) have
routinely been added to consumer products for
several decades in a successful effort to reduce
fire-related injury and property damage.1) The
five major BFRs in use are tetrabromo bisphenol
A (TBBPA), hexabromocyclododecane (HBCD),
and three polybrominated diphenylether (PBDEs)
known as decabromodiphenylether (DBDE),
octabromodiphenylether (OBDE), and pentabromodiphenylether (pentaBDE). More than 200000
metric tons of BFRs are produced worldwide each
year. BFR production has increased dramatically
∗1
Present address: Laboratory of Veterinary Pathology, Tokyo
University of Agriculture and Technology, 3–5–8 Saiwai-cho,
Fuchu, Tokyo 183–8509, Japan
∗
To whom correspondence should be addressed: Division
of Biochemistry and Immunochemistry, National Institute of
Health Sciences, 1–18–1 Kamiyoga, Setagaya-ku, Tokyo 158–
8501, Japan. Tel.: +81-3-3700-1141 (Ext. 243); Fax: +81-33707-6950; E-mail: [email protected]
over the past 20 years. Of the 117950 tons of BFRs
consumed in Asia in 2001, approximately 76% was
TBBPA, 21% PBDEs, and 3% HBCD.2)
DBDE is the major PBDE product in all markets, and accounts for approximately 80% of total
PBDE production worldwide.3) DBDE is used as an
additive flame retardant primarily in electrical and
electronic equipment, as well as in textiles, where
it is applied as a polymer backcoat to fabric. Limited evaluations of the ecologic effects of PBDEs
have been conducted. In general, the lower brominated mixtures are more toxic than the higher congeners. As PBDEs are lipophilic and easily bioaccumlated in organisms through the food chain, PBDEs have recently been detected in many marine
products (average concentration of PBDEs in raw
fish, 0.22 ng/g),4) and exponential increases in the
concentration of PBDE in human milk (0.07 ng/g
lipid in 1972 and 4.02 ng/g lipid in 1997) have been
reported.5)
Mammalian toxicity studies have been conducted in both rats and mice. The most extensive
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No. 4
data set exists for DBDE, with studies ranging from
acute to chronic laboratory studies. The US National Toxicology Program (NTP) conducted 2-year
feeding studies of DBDE6) and showed that high
doses up to 50000 ppm in the diet resulted in neoplastic nodules in the liver in both male and female
rats. DBDE had similar effects on male mice but
did not induce neoplastic nodules in female mice.
Few effects other than the low incidence of tumors
were seen. A developmental neurotoxic effect of
pentaBDE (BDE99) in mice7) and a thyroid systemimpairing effect of commercial tetra and penta BDE
mixtures in several experimental models8, 9) have
been reported. However, little information exists
on the reproductive effects of DBDE. Furthermore,
regulation of the developing immune system by thyroid hormone10, 11) and the effects of experimentally
induced hypothyroidism in adult mice on the immune response have been reported,12) although the
developmental effects of DBDE on the immune system have not yet been examined. We therefore investigated the effects of DBDE on the developing
immune system in rats to determine the safe dose of
the compound.
MATERIALS AND METHODS
Chemicals and Animals —— DBDE was purchased from Wako Pure Chemical Industries
(Osaka, Japan). Pregnant Sprague-Dawley: IGS
female rats were purchased from Charles River
Japan, Inc. (Kanagawa, Japan) on gestational day
3 (GD3). The animals were housed individually
in polycarbonate cages (SK-Clean, 41.5 cm × 26 cm
× 17.5 cm; CLEA Japan Inc., Tokyo, Japan) on
wood chip bedding (Soft Chip; Sankyo Lab Service Corp., Tokyo, Japan) and maintained in an
air-conditioned animal room (temperature 24 ± 1◦ C,
relative humidity 55 ± 5%) on a 12-hr light/dark cycle. They were given ad libitum access to food and
tap water. CRF-1, a regular rodent diet, obtained
from Oriental Yeast Co. Ltd. (Tokyo, Japan) was
used as the basic diet for the offspring, while dams
from GD10 to PND 21 received a soy-free diet (Oriental Yeast Co. Ltd.), prepared based on the NIH-07
open-formula rodent diet and with nutritional standards the same as those of the the CRF-1 (supplier’s
analysis).13)
Experimental Protocol —— Immediately after arrival at the test facility, dams were provided with
a powdered soy-free diet. On GD10, they were
randomized into four groups (6–8 dams/group) and
provided with a soy-free diet that contained DBDE
at a concentrations of 0, 10, 100, or 1000 ppm until PND 21. Based on the results of a preliminary
study, the highest dose was selected as the level
to maintain pregnancy, delivery, and lactation (data
not shown). On PND 2, the number, weight, and
anogenital distance (AGD) of the neonates were
recorded. On PND 21, dosing was terminated, the
offspring were weaned, and 10 males and 10 females (at least 1 male and 1 female per litter) per
group were selcted for prepubertal necropsy, and 10
males and 10 females (at least 1 male and 1 female
per litter) per group for necropsy on PND 77. The
diet was changed to CRF-1 at weaning to eliminate
possible modifications due to the long-term use of a
soy-free diet on development after weaning.14)
The prepubertal necropsy of the male rats was
performed on PND 21 to evaluate the weights and
histopathology of immune organs (see details below). On PND 77, offspring were subjected to organ
weight measurements and histopathologic examination of immune-related organs.
All animals were sacrificed by exsanguination
from the abdominal aorta under deep anesthesia.
The animal protocols were reviewed in terms of animal welfare and approved by the Animal Care and
Use Committee of the National Institute of Health
Sciences, Japan.
Cell Phenotyping —— Cell phenotypes were determined using flow cytometric (FCM) analysis
with monoclonal murine antibodies. Two-color or
three-color analysis of spleen, thymus, and peripheral blood subsets was performed. The antibodies used for FCM were phycoerythrin (PE)-labeled
anti-rat CD8a (OX-8; BD Pharmingen), PE-Cy5labeled anti-rat CD4 (OX-35; BD Pharmingen),
fluorescein isothiocyanate (FITC)-labeled anti-rat
CD3 (1F4; BD Pharmingen), PE-labeled antirat CD25 (IL2Rα chain) (OX-39; BD Pharmingen), PE-Cy5-labeled anti-rat CD45RA (OX33; BD
Pharmingen), PE-labeled anti-rat CD71 (transferrin receptor) (OX26; BD Pharmingen), and FITClabeled anti-rat CD161a (NKRP1A) (10/78; BD
Pharmingen) antibodies. All incubations were performed in the dark. A single-cell suspension of lymphocytes in PharMingen Stain Buffer containing 2%
Fetal Bovine serum (FBS) was incubated with 50 µl
of properly diluted monoclonal antibody at 4◦ C for
30 min. The cells were washed by centrifugation
in phosphate buffered saline (PBS) containing 2%
FBS, and after staining, a total of at least 10000 cells
384
was analyzed with a FACS Calibur (Becton Dickinson, Sunnyvale, CA, U.S.A.). The data were analyzed with Cellquest software.
Antibody Production —— Female offspring exposed perinatally to DBDE until weaning (PND 21)
were immunized with keyhole limpet hemocyanin
(KLH) (Calbiochem, 25 µg) plus 1 mg of alum three
times, on PND 23, 33, and 43. Serum was obtained
on PND 50. The serum titers (reciprocal of serum
dilution with colorimetric intensity at 50% of the
maximum level) of KLH-specific IgG and IgM were
determined. A 50-µl volume of KLH (40 µg/ml)
in sodium carbonate buffer 50 mM, pH 9.6, was
added to each well of a 96-well microtiter plate, and
the plate was incubated overnight at 4◦ C. The solutions were discarded, and each well was washed 4
times with PBS 200 µl containing 0.05% Tween 20
(PBS/Tween). To minimize the nonspecific binding
of serum proteins to unoccupied solid-phase sites,
200 µl of 0.1% casein in PBS was added, and the
plates were incubated for 1 hr at room temperature.
The casein solution was removed, and each well was
washed in the same manner as above. Fifty microliters of the diluted serum containing KLH-specific
antibodies was added to each well, and the plates
were incubated for 20 hr at 4◦ C. The solution was
removed, and each well was washed. Fifty microliters of rabbit anti-rat IgM or IgG [10−3 dilution in
PBS containing 0.1% casein (Nordic Immunology,
Tilburg, the Netherlands)] was added to each well,
and the plates were incubated for 1 hr at room temperature. The solution in each well was removed
and washed. Fifty microliter peroxidase-conjugated
donkey anti-rabbit Ig antibodies (1 : 1000, Amersham) in 0.1% casein-PBS was added and incubated
for 1 hr at room temperature and then reacted with a
colorimetric substrate solution (TMB reagent, Cat.
No. 555214; BD Biosciences, San Diego, CA,
U.S.A.). Colorimetric intensity (OD450 ) was measured according to the manufacturer’s protocol.
Hematological Examination —— The red blood
cell (RBC) count, mean corpuscular volume
(MCV), mean corpuscular hemoglobin (MCH),
mean corpuscular hemoglobin concentration
(MCHC), and white blood cell (WBC) count were
determined with an autohematology analyzer (M2000, Sysmex Corp., Kobe, Japan). The percentage
of segmented neutrophils (Seg Neut), banded
neutrophils (Band Neut), eosinophils (Eosino), basophils (Baso), lymphocytes (Lymph), monocytes
(Mono), and erythroblasts (Ebl) among WBCs were
determined with an automated blood cell analyzer
Vol. 54 (2008)
(Microx Heg-120A, Tateishi Electronic Corp.,
Kyoto, Japan). T3, T4, and thyroid stimulating
hormone (TSH) in the sera were measured at the
SRL Corporation (Tokyo, Japan).
Histopathological
Examination —— Immunerelated organs (spleen and thymus) obtained from
each animal and fixed in neutralized formalin
as described above were embedded in paraffin,
processed, and stained with hematoxylin and
eosin (HE). The HE-stained sections were examined under a light microscope and evaluated
histopathologically.
Statistical Analysis —— All values are expressed
as mean ± standard deviation. The group data were
first tested for homogeneity of variance. Groups
were then compared using one-way analysis of variance (ANOVA) and Dunnett’s test. For histopathologic analysis, Fisher’s exact probability test was
used to compare the differences between groups.
RESULTS
Body, Thymus, Spleen and Liver Weights and
Hematologic Examination
The indirect DBDE exposure method, i.e., via
the placenta or maternal milk, was utilized in this
study. On PND 21, no significant differences in absolute body weight were found between male pups
exposed to DBDE and the control male pups, and
8 weeks after the conclusion of DBDE exposure
(PND 77), the 10- to100-ppm DBDE-treated groups
were examined for increases in body weight. No
significant differences in thymus or spleen weight
were found between the DBDE groups and control group, but on PND 21 liver weight was significantly increased in the 10- to1000-ppm DBDE
groups, but their weight had returned to the control level at 8 weeks after DBDE was removed from
the diet (PND 77) (Table 1). An increase in the
liver weight of female rats was also observed on
PND 21 (data not shown). No significant differences in leukocyte numbers were found in the thymus or spleen (data not shown). No significant differences in RBC, MCV, MCH, MCHC, or WBC and
the percentage of neutrophils (Seg and Band Neut),
Eosino, Baso, Lympho, or Mono were found between the male DBDE groups and the control group.
A tendency toward an increase in the percentage of
Ebl was observed in the DBDE group on PND 21
(data not shown).
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No. 4
Table 1. Body and Organ Weights of Offspring Exposed to DBDE during the Period from Mid-Gestation to Lactation
0
PND 21
No. of offspring examined
BW (g)
Liver (g/ 100 g BW)
Spleen (g/ 100 g BW)
Thymus (g/ 100 g BW)
PND 77
No. of offspring examined
BW (g)
Liver (g/ 100 g BW)
Spleen (g/ 100 g BW)
Thymus (g/ 100 g BW)
10
51.6 ±
3.62 ±
0.392 ±
0.442 ±
10
10
55.8 ±
3.98 ±
0.417 ±
0.440 ±
6.2 a)
0.26
0.06
0.066
10
414.4 ± 22.3 a)
3.66 ± 0.18
0.191 ± 0.023
0.131 ± 0.017
DBDE in diet (ppm)
100
10
52.7 ±
3.90 ±
0.381 ±
0.411 ±
4.0
0.12 ∗
0.074
0.055
10
447.8 ± 24.1 ∗
3.65 ± 0.12
0.197 ± 0.042
0.121 ± 0.021
a) Mean ± S.D. DBDE, decabromodiphenylether; PND, postnatal day.
(∗ p < 0.05, ∗∗ p < 0.01).
1000
6.0
0.29 ∗
0.088
0.056
10
455.1 ± 22.9 ∗∗
3.62 ± 0.10
0.191 ± 0.035
0.122 ± 0.014
∗, ∗∗ Significantly
10
54.0 ±
4.39 ±
0.382 ±
0.414 ±
3.0
0.27 ∗∗
0.044
0.064
10
423.2 ± 34.5
3.42 ± 0.29
0.189 ± 0.021
0.126 ± 0.031
different from the controls by Dunnett’s test
Table 2. Serum Levels of Thyroid-Related Hormones of the Offspring Exposed to DBDE during the Period
from Mid-Gestation to Lactation
0
PND 21
No. of offspring examined
T3 (ng/ml)
T4 (µg/dl)
TSH (ng/ml)
PND 77
No. of offspring examined
T3 (ng/ml)
T4 (µg/dl)
TSH (ng/ml)
DBDE in diet (ppm)
10
100
1000
10
1.39 ± 0.11 a)
5.19 ± 0.74
5.38 ± 0.89
10
1.35 ± 0.15
4.89 ± 0.84
5.12 ± 0.71
10
1.33 ± 0.18
5.66 ± 0.71
5.85 ± 1.22
10
0.99 ± 0.09
6.02 ± 0.70
8.30 ± 3.40
10
1.01 ± 0.08
6.00 ± 0.66
8.81 ± 1.63
10
1.01 ± 0.11
5.98 ± 0.94
9.71 ± 3.45
a) Mean ± S.D. DBDE, decabromodiphenylether; PND, postnatal day.
by Dunnett’s test (∗ p < 0.05, ∗∗ p < 0.01).
Serum Levels of Thyroid-Related Hormones
As shown in Table 2, on PND 21 the serum level
of the thyroid-related hormone T3 was significantly
decreased in the 1000-ppm DBDE group. On PND
77, the serum T4 level was significantly decreased
in the 1000-ppm DBDE group, and a tendency toward an increase in TSH was also found.
Splenocyte, Thymocyte, and Peripheral Blood
Lymph Subpopulations
The effect of DBDE on the surface phenotype
of Lymph is shown in Table 3. Significant differences were observed in some Lymph populations in
the spleen and peripheral blood cells. On PND 21,
the proportions of splenic CD4+ T cells in the 10ppm group, activated B (CD45RA+CD71+) cells
in 100- to 1000-ppm groups, and activated T cells
∗, ∗∗ Significantly
10
1.17 ± 0.10 ∗∗
4.89 ± 0.54
4.74 ± 0.69
10
1.02 ± 0.11
5.17 ± 0.57 ∗
10.47 ± 2.35
different from the controls
(CD3+CD71+) in the 1000-ppm group were significantly decreased, and on PND 21 and 77 the population of peripheral CD161+ natural killer (NK) cells
was decreased in the 100- to 1000-ppm group. On
PND 77, about 40% of peripheral blood CD161+
NK cells were CD161 and CD4 double-positive
cells. On PND 77, a tendency for the population
of splenic CD161+NK cells to be reduced was also
observed (data not shown).
The CD161 and CD4 double-positive cells
seemed to be CD4+NKT cells,11, 15) and the
CD161+CD4-cells seemed to be classic NK cells.
Figure 1 shows the typical pattern of expression
of CD161 on the peripheral blood Lymph derived
from the DBDE group on PND 77. M1 shows the
CD161-positive portion of the Lymph. Two populations of CD161 positive-Lymph are shown. The
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Vol. 54 (2008)
Table 3. Flow Cytometric Analysis of Lymphocytes of Offspring Perinatally Exposed to DBDE
T cell subpopulations
DBDE:
CD8a(−) CD4(+)
CD8a(+) CD4(−)
CD3(+) CD4(+)
DBDE:
CD8a(−) CD4(+)
CD8a(+) CD4(−)
CD3(+) CD4(+)
Activation of T/B cells
DBDE:
CD3(+) CD71(+)
CD71(+) CD45RA(+)
CD3(−) CD45RA(+)
CD3(+) CD45RA(−)
DBDE:
CD3(+) CD71(+)
CD71(+) CD45RA(+)
CD3(−) CD45RA(+)
CD3(+) CD45RA(−)
Treg, NK, NKT cells
DBDE:
CD25(+) CD4(+)
NKRP1A(+) CD4(+)
NKRP1A(+) CD4(−)
DBDE:
CD25(+) CD4(+)
NKRP1A(+) CD4(+)
NKRP1A(+) CD4(−)
0 ppm
14.98 ± 2.43
6.99 ± 1.49
6.28 ± 1.57
0 ppm
27.99 ± 11.13
18.99 ± 3.73
20.26 ± 4.12
0 ppm
0.51 ± 0.1
3.92 ± 1.12
50.67 ± 10.11
15.21 ± 3.65
0 ppm
0.36 ± 0.12
1.13 ± 0.41
31.13 ± 5.79
50.35 ± 7.72
0 ppm
2.33 ± 0.66
17.04 ± 3.84
6.07 ± 1.37
0 ppm
3.11 ± 0.65
9.95 ± 2.61
12.75 ± 2.55
Values are mean ± S.D. [% (gated)] n = 10.
respectively.
Spleen PND 21
10 ppm
100 ppm
∗
12.25 ± 1.61
12.59 ± 3.08
5.28 ± 0.92 ∗
6.91 ± 1.62
4.68 ± 0.86 ∗
6.27 ± 2.53
Spleen PND 77
10 ppm
100 ppm
21.62 ± 3.01
23.33 ± 3.07
18.02 ± 2.05
18.27 ± 3.29
19.1 ± 3.13
20.94 ± 2.98
Spleen PND 21
10 ppm
100 ppm
0.47 ± 0.14
0.42 ± 0.13
3.16 ± 0.73
2.69 ± 0.84 ∗
49.05 ± 7.13
51.82 ± 7.32
11.88 ± 2.21 ∗
14.83 ± 4.41
Spleen PND 77
10 ppm
100 ppm
0.31 ± 0.09
0.39 ± 0.19
1.09 ± 0.4
1.29 ± 0.46
34.46 ± 2.97
33.83 ± 7.5
46.42 ± 4.09
49.51 ± 6.3
Peripheral blood PND 21
10 ppm
100 ppm
2.58 ± 0.79
2.32 ± 0.74
12.14 ± 5.9
10.92 ± 4.62 ∗
5.48 ± 1.69
5.07 ± 1.21
Peripheral blood PND 77
10 ppm
100 ppm
2.9 ± 0.6
2.44 ± 0.52 ∗
7.37 ± 2.59
7.86 ± 2.38
12.1 ± 3.03
12.62 ± 2.62
∗, ∗∗ Significant
1000 ppm
11.34 ± 2.41 ∗∗
6.73 ± 1.41
4.67 ± 1.53
1000 ppm
21.9 ± 3.66
17.74 ± 1.63
19.02 ± 3.92
1000 ppm
0.38 ± 0.09 ∗
2.42 ± 0.72 ∗∗
51.54 ± 8.79
12.24 ± 2.23
1000 ppm
0.34 ± 0.13
1.15 ± 0.38
36.62 ± 3.12 ∗
46.32 ± 4.11
1000 ppm
1.91 ± 0.68
13.66 ± 4.16
5.51 ± 1.06
1000 ppm
2.28 ± 0.52 ∗
7.02 ± 1.38 ∗
9.12 ± 0.85 ∗∗
difference from control at p < 0.05 and p < 0.01,
Fig. 1. Expression of CD161 (NKRPIA) on the Peripheral Blood Lymphocytes on PND 77 from DBDE-Exposed Pups
On PND 77, the peripheral blood lymphocytes of DBDE-exposed pups were examined for the presence of CD161-positive cells by flowcytometry
as described in the Materials and Methods section. The ordinate shows the relative cell number of cells, and the abscissa shows the log of their
fluorescence intensity.
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No. 4
CD161-high expression population was CD4 negative, and the CD161-moderate expression population was CD4 positive (data not shown). A dosedependent decrease in the percentage of both populations of peripheral CD161-positive Lymph (both
NK cells and CD4+NKT cells) was observed. The
DBDE group examined on PND 77 showed a decrease in CD4+CD25+ regulatory T (Treg) cells
among peripheral blood cells.
Production of Antibody to KLH
Figure 2 shows the serum KLH-specific IgG
titers. ELISA titers of specific IgG to KLH tended to
be enhanced and reduced in the 10-ppm and 1000ppm DBDE group, respectively, but the difference
was not statistically significant. No tendency for a
decrease was observed in the production of KLHspecific IgM.
Histopathology of the Spleen and Thymus
Table 4 shows the histopathology of the thymus
and spleen of SD rats exposed to DBDE perinatally.
On PND 21, a slight reduction in the area of red pulp
and erythropoiesis was observed in some pups in the
100-ppm group. On PND 77, a slight reduction in
the cortical area of the thymus and a reduction in the
marginal region of white pulp and the area of red
pulp in the spleen was observed in some pups in the
1000-ppm group. However, none of the pathologic
changes observed in the DBDE group were significant when compared with the control group.
DISCUSSION
Fig. 2. Effects of DBDE on Production of IgG to KLH
Perinatally DBDE-exposed female pups were immunized with
KLH three times over a 4- to 7-week period. Serum was obtained 1
week after final immunization, and the IgG titer to KLH was measured
using ELISA as described in the Materials and Methods section. Open
circles represent individual values, and dashes interconnected by a line
indicate mean values.
There have been few reports on the effects of
BFRs on the immune system. Although an inhibitory effect of TBBPA on Lymph proliferation
and expression of CD25 in vitro has been reported,
16) there have been few reports on the effects of
other BFRs on the immune system. In this study, we
first examined the effects of a typical BFR, DBDE,
on the developing immune system in vivo.
A developmental neurotoxic effect of pentaBDE
in mice7) and a thyroid system-impairing effect of
commercial tetra and penta BDE mixtures in rats
and mice8, 9) have been reported. Our own exper-
Table 4. Histopathology of the Thymus and Spleen of Male Rats Perinatally Exposed to DBDE
0
PND 21
No. of animals examined
Thymus
Abnormalities detected
Spleen
Reduction of the area of red pulp (±) a)
Reduction of erythropoiesis (±)
PND 77
No. of animals examined
Thymus
Reduction of cortical area (±)
Spleen
Reduction of the mar ginal region of white pulp (±)
Reduction of the area of red pulp (±)
DBDE in diet (ppm)
10
100
1000
10
10
10
10
0
0
0
0
0 b)
0
0
0
3
3
0
0
10
10
10
10
1
0
0
2
0
0
0
0
0
1
1
2
a) Grade of change: ±, minimal. b) Total no. of animals with each finding.
388
iments revealed a decrease in the serum level of
the thyroid hormones T3 (1000 ppm at PND 21)
and T4 (1000 ppm at PND 77) in DBDE-exposed
pups (Table 2). Therefore indirect exposure of the
pups to DBDE through the dams confirmed a thyroid system-impairing effect on pups.
The results in regard to the developing immune
system showed that on PND 21 the proportions of
splenic CD4+ T cells in the 10-ppm group, activated
B (CD45RA+CD71+) cells in the 100- to 1000ppm groups, and activated T cells (CD3+CD71+) in
the 1000-ppm group were significantly decreased,
and on PND 21 and 77 the population of peripheral CD161+NK cells had decreased in the 100- and
1000-ppm groups (Table 3). A tendency toward a
decrease in anti-KLH IgG antibody production was
observed in the 1000-ppm group that was immunized with KLH 3 times (Fig. 2). Since KLH is a Tcell-dependent antigen,17) the reduction in antibody
production seems to be related to the decrease in the
percentage of CD4+ helper T cells or the decrease in
activated B and T cells. These results suggest that
developmental exposure to DBDE had a weak immunomodulatory effect at the highest dosage during
the exposure period, which seems to be correlated
with the perturbation of thyroid hormone homeostasis. Although most of the modulatory effect had recovered to normal levels after growth, a decreasing
effect on the proportion of NK cells remained.
We showed the existence of two populations of
NK cells, CD161 single-positive and CD4/CD161
double-positive cells, in peripheral blood (Fig. 1).
The CD161+CD4 cells seemed to be classic NK
cells, which are a form of cytotoxic Lymph constituting a major component of the innate immune
system for the host rejection of both tumors and
virally infected cells. The CD4/CD161 doublepositive cells seemed to be CD4-positive NKT cells,
known as T cells expressing NK receptors and capable of secreting IL-4 and IFN-γ upon stimulation.18)
Collabolation of NKT cell and Treg (CD4+CD25+)
cells in the prevention of autoimmunity has also
been reported.19) Therefore the decrease in the population of NK cells might have a major influence on
the balance of a variety of immune responses, both
innate and aquired.
Induction of UDP-glucuronide transferase
(UDPGT), the key phase II metabolizing enzyme
involved in the conjugation of T4, has been reported
in commercial PBDE mixture-exposed weanling
rats9) and developmentally exposed (GD6-PND 21)
rats.20) Therefore induction of UDPGT presumably
Vol. 54 (2008)
also occurred in our DBDE-exposed pups.
DBDE has historically been shown to be poorly
absorbed after oral exposure.21) However, recent
studies in the rat have shown that DBDE can be
absorbed (> 10% of the dose) when administered
orally, and that higher concentrations are found in
the plasma and highly perfused tissues such as the
liver, heart, adrenal gland, and kidney, whereas adipose tissue has lower concentrations.22) The same
group of investigators also showed that approximately 10% of the dose was eliminated in the bile
as hydoxy/methoxy metabolites containing five to
seven bromide atoms. It seems to be reasonable
that lipophilic DBDE could pass through dam rat
placenta or be secreted in maternal milk and exert
its immunomodulative activities in the offspring, although the possibility that the drug indirectly affects
the offspring through decreases in the maternal thyroid hormones is not completely excluded. Shinohara et al. reported that a 4-hydroxy group is necessary for estrogen and thyroid hormone-disrupting
activities, and 3,5-dibromo substitution is also necessary for thyroid hormone-disrupting activity.23)
Since the concentrations of these metabolites of
DBDE in orally fed rats seems to be low, their thyroid hormone-disrupting effect would also seem to
be slight.
In conclusion, developmental exposure to
DBDE seems to have a weak immunomodulatory
effect at the highest dosage, resulting in a decrease
in NK cell populations, a decrease in the helper T
cell population, and a decrease in activated B and T
cells, which seems to be correlated with the perturbation of thyroid hormone homeostasis.
Acknowledgement This study was supported by
a grant from the Ministry of Health, Labor and Welfare of Japan.
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